The focus of this proposal is to define the role of DNA methylation in development and cancer. We will investigate the influence of DNA methylation on the expression of determination and transforming genes and study interactions between them, determine how methylation patterns evolve during tumor progression and test the hypothesis that DNA methylation can skew the genetics of some human cancers. Cells of the mouse embryo cell line 10T1/2 primed to differentiate with 5-azacytidine, will be used to investigate hierarchies of gene control in mesenchymal determination and differentiation. I am particularly interested in defining the role of methylcytosine in this process. The influence of cellular determination on oncogenesis and the interplay between transforming and determination genes will ge investigated in the same system. The potential for DNA methylation to silence transforming genes will be examined by the molecular cloning and characterization of a novel oncogene in human osteogenic sarcoma cells treated with 5- azadeoxycytidine. Changes in methylation levels and patterns during tumor progression will be tracked in fresh samples of human bladder tumors. The possibility that allele specific methylation of human genes can skew the genetics of human cancer, due to the inherent mutagenic activity of 5-methylcytosine, will be examined in human retinoblastoma and Wilms' tumor.